The objective of the proposed research is to establish the quaternary structure and mechanism of action of the enzyme hydrogenase. This is being studied by optical and EPR spectra of the pure enzyme on reaction with the substrate, H2, and with the inhibitors, O2 and CO. A second objective is to study the regulation of hydrogenase activity in enterobacteria to determine the role of the enzyme in microbial physiology, define conditions for its induced synthesis at high levels, mapping and transferring the hydrogenase gene, and isolation of mutant forms of the enzyme with altered properties. Mutagenesis followed by selective growth conditions and penicillin treatment will be used to isolate hydrogenase-negative cells from hydrogenase-positive parents. The mutation will be characterized and these cells will be used as recipients for the hydrogenase gene from positive cells. The same general approach will be used to isolate mutant cells which contain an oxygen-insensitive hydrogenase which retains full catalytic activity in the presence of oxygen.